Ebola virus (EBOV) can cross cellular barriers, establish persistence, and re-emerge in survivors months later in the eye, central nervous system, and semen, leading to long term pathology and sequelae. Indeed, a wide spectrum of ocular complications, including hemorrhages, blindness, and uveitis, have been reported following both acute EBOV infection and during long-term convalescent stages. The mechanisms by which filoviruses enter, persist, and re-emerge in the eye remain unknown. Retinal pigment epithelial (RPE) cells are permissive for EBOV infection and are thought to serve as the intraocular reservoir for persistence and spread of EBOV. Based on our preliminary data, we hypothesize that EBOV VP40 (eVP40) contributes to the spread of EBOV to the human eye by interacting with tight junctional (TJ) complex proteins in the RPE and/or corneal endothelium, thereby weakening these barriers to facilitate virus infection and spread during acute infection, as well as during re-emergence from a persistent state. Specifically, we have shown that the PPxY motif conserved in filovirus VP40 proteins can interact with select host WW-domain bearing proteins that are involved in maintaining and regulating the physical and functional integrity of cellular TJs. These VP40 interactors include MAGI3, a member of the MAGUK superfamily of proteins, that functions as an adaptor/scaffolding protein to maintain junction integrity and barrier function as well as YAP and TAZ; paralogues and downstream effectors of the Hippo signaling pathway that regulate genes involved in cell proliferation and migration and junctional integrity. The interaction of YAP/TAZ and MAGI3 with the PPxY motifs of filovirus VP40 is particularly intriguing, as these host proteins are also regulated by the binding of their WW-domains to the N-terminal PPxY motifs of the host protein AMOT (Angiomotin). Indeed, Amot negatively regulates YAP activity by binding and sequestering YAP at TJs, and promotes TJ assembly and integrity by interacting with the MAGI family of scaffolding proteins. Since Amot, via its PPxY motifs, appears to be a ?master regulator? of TJ formation and function, we hypothesize that the PPxY motifs of filovirus VP40 may compete with those of Amot to disrupt TJ formation and function in virus infected cells, thereby enhancing cellular permeability and virus spread. In this high risk, high impact proposal, we will use a powerful and innovative eye-on-a-chip technology to provide a human cell-based model system with an unprecedented level of physiological realism to investigate: 1) the role of VP40 in altering the most essential barriers of the human eye, and 2) to determine whether our previously validated PPxY inhibitors not only can block VP40-mediated egress and spread, but also prevent VP40-mediated disruption of TJ barriers and disease progression in these eye models. This exploratory and innovative approach will provide new fundamental insights into the molecular mechanisms of filoviral-host interactions in the human eye that may contribute to transmission and pathogenesis of these deadly viruses, and may identify a new therapeutic strategy to treat filovirus infections in these ocular tissues.